The metabolism of ethylbenzene and styrene to mandelic acid: stereochemical considerations

1. The stereochemistry of mandelic acid, produced as a major urinary metabolite of ethylbenzene and styrene in rat and man has been investigated. Although these solvents are both achiral they are metabolized to chiral metabolites, via a series of chiral intermediates. 2. Analytical methods (g.l.c.-mass spectrometry, h.p.l.c. and 19F-n.m.r.) have been developed for the determination of the enantiomeric composition of mandelic acid in urine. 3. These methods have been applied to the study of the metabolic stereochemistry of ethylbenzene and styrene in rats dosed orally (100 mg/kg body weight) and in human volunteers exposed to atmospheres containing these solvents at the upper limits prescribed for workplaces by the UK Health and Safety Executive (100 ppm in air). 4. Results show that whereas only the R-enantiomer of mandelic acid was excreted after ethylbenzene exposure, the mandelic acid formed from styrene was essentially racemic. In three workers occupationally exposed to styrene, ratios of R to S isomers of 1.16, 1.27 and 1.14 were found. A synthetic R/S mixture of mandelic acid had an R/S ratio of 1.03. 5. The implications of these findings for the biological monitoring of workers occupationally exposed to stryrene and/or ethylbenzene are discussed.     

Styrene-oxide N-terminal valine haemoglobin adducts in reinforced plastic workers: possible influence of genetic polymorphism of drug-metabolising enzymes

Styrene is one of the most important organic chemicals used worldwide. In humans, styrene metabolism involves oxidation by cytochrome P450 monooxygenases (CYPs) to styrene-7,8-oxide, an epoxide thought to be responsible for the genotoxic effects of styrene exposure, and detoxification by means of epoxide hydrolase (mEH) and glutathione S-transferases (GSTs). The objective of this study was to investigate if genetic polymorphisms of metabolic enzymes modulate the level of urinary styrene metabolites and styrene oxide adducts with N-terminal valine of human globin (SO-Hb) in 75 workers occupationally exposed to styrene and 77 unexposed controls. The mean air concentration of styrene in the breathing zone of workers (30.4ppm) was higher than the threshold limit value of 20ppm recommended by the American Conference of Governmental Industrial Hygienists (ACGIH), and the biological exposure index adopted by the ACGIH for exposure to styrene prior to the next shift (MA+PGA=400mg/g creatinine) was exceeded, indicating that styrene exposure for this group of workers was higher than recommended. A highly significant correlation was observed between styrene concentration in the breathing zone and the MA+PGA in urine of workers (r=0.85, P<0.001). The levels of SO-Hb adducts in exposed workers were significantly increased as compared with controls, although no difference was observed between subjects stratified as high and medium exposure categories based on MA+PGA excretion. Regarding the effect of the genetic polymorphisms we found that the level of SO-Hb adducts might be modulated by the predicted mEH enzymatic activity in the exposed workers. From our data we conclude that SO-Hb adduct measurement is a complementary method to MA+PG measurement for assessing exposure to styrene at occupational and environmental levels, which reflects a more extensive exposure period.     

Estimation of the dermal absorption of m-xylene vapor in humans using breath sampling and physiologically based pharmacokinetic analysis.

A physiologically-based pharmacokinetic model, containing a skin compartment, was derived and used to simulate experimentally determined exposure to m-xylene, using human volunteers exposed under controlled conditions. Biological monitoring was conducted by sampling, in exhaled alveolar air and blood, m-xylene and urinary methyl hippuric acid concentrations. The dermal absorption of m-xylene vapor was successfully and conveniently studied using a breath sampling technique, and the contribution to m-xylene body burden from the dermal route of exposure was estimated to be 1.8%. The model was used to investigate the protection afforded by an air-fed, half-face mask. By iteratively changing the dermal exposure concentration, it was possible to predict the ambient concentration that was required to deliver the observed urinary excretion of methylhippuric acid, during and following inhalation exposure to 50 ppm m-xylene vapor. This latter extrapolation demonstrates how physiologically-based pharmacokinetic modeling can be applied in a practical and occupationally relevant way, and permitted a further step not possible with biological monitoring alone. The ability of the model to extrapolate an ambient exposure concentration was dependent upon human metabolism data, thereby demonstrating the mechanistic toxicological basis of model output. The methyl hydroxylation of m-xylene is catalyzed by the hepatic mixed function oxidase enzyme, cytochrome P450 2E1 and is active in the occupationally relevant, (<100 ppm) exposure range of m-xylene. The use of a scaled-up in vitro maximum rate of metabolism (Vmaxc) in the model also demonstrates the increasingly valuable potential utility of biokinetic data determined using alternative, non-animal methods in human chemical-risk assessment.     

Influence of genetic polymorphisms of styrene-metabolizing enzymes and smoking habits on levels of urinary metabolites after occupational exposure to styrene

Here we evaluate the influence of individual genetic polymorphisms of drug-metabolizing enzymes as well as body mass index (BMI) and lifestyle (smoking, alcohol consumption) on urinary metabolites after occupational exposure to styrene. Seventy-three workers exposed to styrene in a reinforced-plastics workplace were studied. The personal styrene exposure in the air and the urinary styrene metabolites mandelic acid and phenylglyoxylic acid were measured. The subjects' genetic polymorphisms in the genes that encode the styrene-metabolizing enzymes CYP2E1, CYP2B6, EPHX1, GSTM1, GSTT1 and GSTP1 were determined. Neither genotype nor lifestyle significantly affected urinary metabolites. There was, however, an interaction between the CYP2E1 genotype and smoking. Among non-smokers, urinary styrene metabolites were significantly decreased in subjects with c1/c1 alleles of CYP2E1 as compared with those with the c1/c2 genotype. There was no significant difference in urinary metabolites among smokers. When the combined influence of the CYP2B6 genotype and the predicted activity of EPHX1 were examined, urinary metabolites in subjects with low enzyme activity were lower than in those with medium or high activity after high styrene exposure (>or=50 ppm). The results suggest that genetic susceptibility and lifestyle should be considered in biological monitoring of exposure to styrene.     

Reproductive toxicology of inhaled styrene oxide in rats and rabbits

Experiments were performed to evaluate reproductive and developmental toxicology in rats and rabbits exposed to styrene oxide by inhalation. Female rats were exposed to 100 or 300 ppm styrene oxide or to filtered air for 7 h/day, 5 days/week for 3 weeks. Extensive mortality occurred in rats that received prolonged exposure to 100 ppm styrene oxide while 300 ppm was rapidly lethal. As a result exposures were terminated in this latter group and the group was eliminated from further study. The rats of the 0 and 100 ppm groups were then mated and exposed to 0 or 100 ppm styrene oxide daily through 18 days of gestation (dg). Female rabbits were artificially inseminated and exposed for 7 h daily to 0, 15, or 50 ppm styrene oxide through 24 dg. Both of these lower concentrations used for exposure of the rabbits produced mortality of does. The rats were killed at 20 dg and the rabbits at 30 dg. Pregnant animals were examined for toxic changes including altered tissue weights and histopathologic effects. Litters were evaluated using several measures of embryotoxicity, and live fetuses were examined for external, visceral, and skeletal malformations. Exposure during gestation appeared to increase preimplantation loss in rats, and tended to increase the incidence of resorptions in rabbits. In both species, fetal weights and crown-rump lengths were reduced by gestational exposure. The incidences of ossification defects of the sternebrae aned occipital bones were increased by gestational exposure of rats to styrene oxide. These results indicate that inhalation exposures at these concentrations produce reproductive and development toxicity, as well as maternal toxicity.     

New approaches to the metabolism of xylenes: verification of the formation of phenylmercapturic acid metabolites of xylenes

It was the aim of this study to ascertain whether xylenes form phenylmercapturic acids via aromatic epoxides in the human metabolism. Aromatic epoxides are suspected to exert mutagenic properties. Therefore we developed an LC/MS/MS procedure for the determination of these mercapturic acids. Using this method we were able to detect dimethylphenylmercapturic acid (DPMA) in urine samples of persons occupationally exposed to xylenes. The unequivocal LC/MS/MS detection of phenylmercapturic acid metabolites of xylene thanks to authentic standards was verified with an independent GC/MS method. Xylene concentrations in the air of the workplaces ranged between 0.7 and 58.1 ppm (median 12.6 ppm). The excretion of methylhippuric acid in the urine samples of the workers (n=27) ranges from 19.8 to 2332.5 mg/l (median 450.9 mg/l). DPMA was detected in only 9 samples of 27 exposed persons. According to a rough calculation DPMA is only formed in a ratio of 0.0003% respective to the xylene main metabolite MHA. That means that even under occupationally relevant xylene exposure potential mutagenic potencies should be negligible. DPMA in urine is not sensitive enough for general biomonitoring purposes due to the low ratio of excretion. MHA should therefore be used for biomonitoring from now on. Furthermore, our results show that irrespective of the structure of xylene isomers there is no preferences in metabolism.     

Styrene-7,8-oxide in blood of workers exposed to styrene

A field study was carried out on 13 workers exposed to styrene vapors at time-weighted average concentrations between 10 and 73 ppm. The reactive intermediate styrene-7,8-oxide was determined in blood samples using a direct gas chromatographic method. Styrene-7,8-oxide concentrations were in the range between 0.9 and 4.1 μg/l blood. Linear correlations were found between styrene-7,8-oxide in blood and styrene in ambient air and blood. For an exposure concentration of 20 ppm styrene (German MAK value) a steady-state level of about 1 μg styrene-7,8-oxide/l blood was calculated. Received: 3 February 1994/Accepted: 7 April 1994     

Genotoxicity of styrene-7,8-oxide and styrene in Fisher 344 rats: a 4-week inhalation study

The cytogenetic alterations in leukocytes and the increased risk for leukemia, lymphoma, or all lymphohematopoietic cancer observed in workers occupationally exposed to styrene have been associated with its hepatic metabolisation into styrene-7,8-oxide, an epoxide which can induce DNA damages. However, it has been observed that styrene-7,8-oxide was also found in the atmosphere of reinforced plastic industries where large amounts of styrene are used. Since the main route of exposure to these compounds is inhalation, in order to gain new insights regarding their systemic genotoxicity, Fisher 344 male rats were exposed in full-body inhalation chambers, 6 h/day, 5 days/week for 4 weeks to styrene-7,8-oxide (25, 50, and 75 ppm) or styrene (75, 300, and 1000 ppm). Then, the induction of micronuclei in circulating reticulocytes and DNA strand breaks in leukocytes using the comet assay was studied at the end of the 3rd and 20th days of exposure. Our results showed that neither styrene nor styrene-7,8-oxide induced a significant increase of the micronucleus frequency in reticulocytes or DNA strand breaks in white blood cells. However, in the presence of the formamidopyridine DNA glycosylase, an enzyme able to recognize and excise DNA at the level of some oxidized DNA bases, a significant increase of DNA damages was observed at the end of the 3rd day of treatment in leukocytes from rats exposed to styrene but not to styrene-7,8-oxide. This experimental design helped to gather new information regarding the systemic genotoxicity of these two chemicals and may be valuable for the risk assessment associated with an occupational exposure to these molecules.     

Occupational exposure to styrene: modulation of cytogenetic damage and levels of urinary metabolites of styrene by polymorphisms in genes CYP2E1, EPHX1, GSTM1, GSTT1 and GSTP1

Styrene is widely used in the production of various plastics, synthetic rubber and resins. The aim of this study was to evaluate if individual polymorphisms in xenobiotic metabolizing enzymes, related with the metabolic fate of styrene, could modify individual susceptibility to the possible genotoxic effects of the styrene exposure. Twenty-eight reinforced plastic workers and 28 control subjects were studied. In the selected population the urinary styrene metabolites mandelic (MA) and phenylglyoxylic (PGA) acids were quantified, sister chromatid exchanges (SCE) and micronuclei (MN) were assessed in peripheral lymphocytes and all the subjects were genotyped for GSTM1, GSTT1 (gene deletions), GSTP1 (codon 105 ile==>val), EPHX1 (codons 113 tyr==>his and 139 his==>arg) and CYP2E1 (DraI polymorphism in intron 6). The results obtained showed a significant difference between the levels of SCE, but not in MN levels, in exposed workers as compared with the control group. The GSTP1 and CYP2E1 individual genotypes modulate the baseline levels of SCE that are lower in non-wild type individuals for both polymorphisms. The GSTM1 null individuals with low levels of exposure have significantly higher urinary levels of MA+PGA. The present data seem to suggest that apart from the methodology usually used for monitoring populations occupationally exposed to styrene (urinary metabolites and biomarkers of early biological effects) the analysis of individual genotypes associated with the metabolic fate of styrene should also be carried out in order to evaluate the individual genetic susceptibility of exposed populations.     

Stereometabolism of styrene in man

Chiral styrene metabolites obtained during initial styrene exposure of test persons were determined in urine samples using capillary gas chromatography. A typical time-dependent urinary concentration profile of one person over a 49-h period is presented and compared with the results of a previous study of occupationally exposed workers and an unexposed control group. Maximum levels of excretion of all styrene metabolites were observed at about the end of a 9-h workshift. Forty hours after exposure, the L/D-ratio of mandelic acid had subsided to the initial value, and the L/D-ratio of phenylethylene glycol to a value equal or slightly above the initial value.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

DNA damage and susceptibility assessment in industrial workers exposed to styrene

Styrene is a widely used chemical in the manufacture of synthetic rubber, resins, polyesters, and plastics. The highest levels of human exposure to styrene occur during the production of reinforced plastic products. The objective of this study was to examine occupational exposure to styrene in a multistage approach, in order to integrate the following endpoints: styrene in workplace air, mandelic and phenylglyoxylic acids (MA + PGA) in urine, sister chromatid exchanges (SCE), micronuclei (MN), DNA damage (comet assay), and genetic polymorphisms of metabolizing enzymes (CYP2E1, EPHX1, GSTM1, GSTT1, and GSTP1). Seventy-five workers from a fiberglass-reinforced plastics factory and 77 unexposed controls took part in the study. The mean air concentration of styrene in the breathing zone of workers (30.4 ppm) and the mean concentration of urinary metabolites (MA + PGA = 443 ± 44 mg/g creatinine) exceeded the threshold limit value (TLV) and the biological exposure index (BEI). Significantly higher SCE frequency rate and DNA damage were observed in exposed workers, but MN frequency was not markedly modified by exposure. With respect to the effect of genetic polymorphisms on different exposure and effect biomarkers studied, an increase in SCE levels with elevated microsomal epoxide hydrolase activity was noted in exposed workers, suggesting a possible exposure-genotype interaction.     

S-p -Toluylmercapturic acid in the urine of workers exposed to toluene: a new biomarker for toluene exposure

A mercapturic acid attached to the aromatic ring of toluene was for the first time detected in human urine as a metabolite of toluene. Since the metabolism of toluene is usually considered to take place at the side-chain, this gives, besides the biosynthesis of cresols, a further hint of a metabolic conversion of the aromatic system. We examined a group of 33 workers occupationally exposed to toluene, determining the concentrations of toluene in ambient air and in whole blood, o-cresol and hippuric acid in urine and p-toluylmercapturic acid (p-TMA) in urine. All blood and urine samples were collected post-shift. The renal excretion of S-p-toluylmercapturic acid showed highly significant correlations with established parameters of a biological monitoring of toluene. The median ambient air concentration was 63 ppm, ranging from 13 to 151 ppm, the median concentration of toluene in whole blood was 804 μg/l, corresponding to median urinary concentrations for o-cresol of 2.3 mg/l, hippuric acid of 2.3 g/l and p-TMA of 20.4 μg/l. p-TMA was not detectable in urine samples of a control group of 10 non-exposed persons. Both the German Biological Tolerance Values (BAT-values) for toluene in blood (1000 μg/l) and o-cresol in urine (3 mg/l) correspond to a mean p-TMA elimination of ∼50 g/l, and thus are in agreement with each other. According to our results p-TMA reflects internal toluene exposure diagnostically sensitive and specifical. With the developed analytical procedure we determined a median benzylmercapturic acid (BMA) concentration of 190 μg/l in the urine samples of the toluene exposed persons. We also determined a median BMA concentration of 30 μg/l in the control samples of non-exposed persons. However, these results are preliminary and require further confirmation as the reliability of the method was determined only for p-TMA. Received: 15 July 1997 / Accepted: 24 September 1997     

Neurobehavioral effects of acute styrene exposure in fiberglass boatbuilders

A field investigation of the effects of acute exposure to styrene among fiberglass boatbuilders was performed. Personal samples of styrene in breathing zone air and postshift urinary mandelic acid were collected for 105 workers exposed and not exposed to styrene in 6 fiberglass boatbuilding companies in New England. Three tests from the computerized Neurobehavioral Evaluation System (NES) were performed by the subjects in the morning before exposure to styrene, near midday, and at the end of the work day. Duration of exposure averaged 2.9 years (SD=4.6), 8-hour TWA styrene exposure averaged 29.9 ppm (SD=36.2), and urinary mandelic acid averaged 347 mg/g creatinine (SD=465). Regression analyses indicated a statistically significant relationship between postshift performance on the Symbol-Digit test and both acute styrene exposure and mandelic acid. Other analyses comparing workers exposed to less than 50 ppm and greater than 50 ppm styrene also showed a significant effect on Symbol-Digit performance. All three NES tests showed test-retest correlation coefficients above .80, and ease of use for collection of neurobehavioral data under field conditions was demonstrated.     

Multicenter field trial on possible health effects of toluene. I. Toluene body burdens in workers of the rotogravure industry

Ambient air toluene concentrations as well as corresponding individual blood toluene levels were measured under conditions of a field trial, as basis for a correlation with possible acute effects. While the results of various psycho-physiological and medical evaluations after acute (Neubert et al., 2001) and long-term toluene exposure (Gericke et al., 2001) are published in accompanying papers, this publication deals with the exposure levels and body burdens characteristic of workers in the rotogravure industry in Germany at the time of the investigation (1993-1995). Besides providing some information on the exposure at various work-areas under occupational conditions, the correlation between a time-weighted average of the ambient air concentration with the corresponding blood toluene levels is analyzed. Limitations of such an attempt and possible pitfalls are discussed. In the largest field study so far performed on toluene exposure, 12 companies of the German rotogravure industry (and a total of 1528 volunteers) participated. Altogether, complete data sets, i.e. on both ambient air as well as blood toluene levels, were obtained from 1244 male and 124 female participants of the rotogravure industry with quite different degrees of toluene exposure. Rotogravure printers and their helpers were exposed to the highest toluene concentrations in ambient air. On the day of the evaluation, of 806 male volunteers within this group (of 1261 with verified exposure in air), 35 were exposed to a time-weighted average of 100 ppm (i.e. 375 mg/m(3)) or above, and 155 of the printers to concentrations between 50 and 100 ppm. Of the remaining 455 male participants of the rotogravure factories ('non-printers and helpers'), only three were exposed to toluene concentrations above 50 ppm. Only one of the 124 women working in the rotogravure factories was exposed to an average toluene concentrations above 100 mg/m(3) (i.e. 27 ppm). In 66 of the male volunteers toluene levels in blood of >850 microg/l were measured and 14 showed levels exceeding 1700 microg/l. When attempting to predict the resulting individual blood toluene levels from measurements of ambient air concentrations under field conditions, a considerable uncertainty is to be expected. We found a correlation coefficient of the regression curve of about 0.70, with numerous outliers (and a variation of the 12 factories between 0.52 and 0.88).     

Assessment of biotransformation of the arene moiety of styrene in volunteers and occupationally exposed workers

Styrene is a chemical widely used in the plastic industry. The main pathway of styrene metabolism in humans occurs via the oxidation to styrene-7,8-oxide (7,8-SO). The aim of this study was the investigation of a minor metabolic route, involving the oxidation of the arene moiety of styrene, by means of the characterization of the conjugated urinary metabolites of 4-vinylphenol (4-VP). 4-vinylphenol-glucuronide (4-VP-G) and -sulfate (4-VP-S), were measured by liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS) from 174 workers belonging to three cohorts recruited in European countries and from 26 volunteers exposed to 50 mg/m(3) (11.8 ppm) of styrene for 8 h. The 4-VP conjugates represented about 0.5-1% of the total excretion of styrene metabolites. Both 4-VP-G and 4-VP-S are eliminated with a monophasic kinetic, the glucuronide being excreted faster (half-time, 2.2 +/- 0.2 h) than the sulfate (half-time 9.7 +/- 1.7 h). The urinary 4-VP was found to be significantly correlated both with airborne styrene (r = 0.607, p < 0.001) and the sum of MA and PGA (r = 0.903, p < 0.001 in "end-of-shift" samples). Apart from 7,8-SO, 4-VP is the only styrene metabolite not shared with ethylbenzene and therefore thought to be a highly specific marker of styrene exposure. However, a measurable background excretion of 4-VP was also found in all urine samples from controls not occupationally exposed to styrene. This background appears to be highly correlated to smoking (p < 0.001) and possibly also to the dietary intake of styrene or 4-VP. Consequently, the use of 4-VP as a biomarker of styrene exposure is recommended for exposures exceeding 1 ppm.     

Cytogenetic analysis using fluorescence in situ hybridization (FISH) to evaluate occupational exposure to carcinogens

Chromosomal aberrations determined by conventional method or fluorescence in situ hybridization (FISH) technique with whole chromosome painting are used as biomarkers of effect. Groups occupationally exposed to 1,3-butadiene (BD), acrylonitrile, ethyl benzene and benzene in petrochemical industry, and carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) from ambient air were followed by conventional method and FISH painting for chromosomes # 1 and # 4, in total 383 subjects, including controls. No effect was observed by either method with exposure to 1,3-butadiene < 1mg/m(3) and acrylonitrile < 0.3mg/m(3). Ethyl benzene and benzene exposure significantly increased chromosomal aberrations by both methods, which decreased after the implementation of preventive measures. The genomic frequency of translocations by FISH calculated as FG/100 was significantly increased in city policemen versus control group exposed to c-PAHs from ambient air (1.72+/-1.57 versus 1.25+/-1.11, P<0.05). The method of FISH with whole chromosome painting seems to be more sensitive to detect chromosomal injury by occupational exposure to carcinogens than conventional method.     

Hearing loss in rats caused by inhalation of mixed xylenes and styrene

We have reported that inhalation exposure of rats to toluene causes permanent hearing loss, e.g. Pryor et al. Neurobehav. Toxicol. Teratol. 5, 53-62 (1983). Therefore, it was of considerable interest to examine the ototoxic potential of two structurally related solvents--mixed xylenes and styrene--compared with that of toluene. Male, weanling Fischer-344 rats were exposed to clean air or solvents in four identical 62.5 l Plexiglas chambers. Exposures to 800, 1000, and 1200 ppm were daily for 14 hours/day and lasted 6 weeks for mixed xylenes, 3 weeks for styrene. An additional experiment with xylenes examined the effect of exposure for only 4 (1700 ppm) or 8 (1450 ppm) hours or for 8 (1450 ppm) hours on three consecutive days to compare the results with those obtained with toluene under comparable exposure schedules. Both xylenes and styrene caused marked hearing loss as assessed by behavioral (conditioned avoidance) and electrophysiologic (brainstem auditory-evoked response) methods. Moreover, both solvents appeared to be more potent ototoxicants than toluene, as indicated by effective concentration, effective durations of exposure, and the range of frequencies affected.     

Neurobehavioral effects of experimental exposures to low levels of styrene

Two experimental studies were conducted with the intention to simulate exposure characteristics of work places with styrene exposure and to investigate the risk for neurobehavioral impairments. In experiment I 16 volunteers (8 in the morning, 8 in the afternoon) were exposed to 0.5 and 20 ppm styrene on a constant level for 3h. In experiment II 24 volunteers (12 in the morning, 12 in the afternoon) were exposed for 4h to 0.5 and 20 ppm styrene on a constant level as well as to a changing exposure between 0.5 and 40 ppm with a TWA of 14 ppm. Simple reaction, choice reaction, attention, acute symptoms, and ratings for well-being were measured. Exposure related performance effects could not be detected. However, 6h time change resulted in delayed choice reactions in the morning hours. Analysing acute symptoms and the state of well-being the impact of styrene did not reach adverse extents of impaired well-being.     

Risk assessments for chronic exposure of children and prospective parents to ethylbenzene (CAS No. 100-41-4)

Potential chronic health risks for children and prospective parents exposed to ethylbenzene were evaluated in response to the Voluntary Children's Chemical Evaluation Program. Ethylbenzene exposure was found to be predominately via inhalation with recent data demonstrating continuing decreases in releases and both outdoor and indoor concentrations over the past several decades. The proportion of ethylbenzene in ambient air that is attributable to the ethylbenzene/styrene chain of commerce appears to be relatively very small, less than 0.1% based on recent relative emission estimates. Toxicity reference values were derived from the available data, with physiologically based pharmacokinetic models and benchmark dose methods used to assess dose–response relationships. An inhalation non-cancer reference concentration or RfC of 0.3 parts per million (ppm) was derived based on ototoxicity. Similarly, an oral non-cancer reference dose or RfD of 0.5 mg/kg body weight/day was derived based on liver effects. For the cancer assessment, emphasis was placed upon mode of action information. Three of four rodent tumor types were determined not to be relevant to human health. A cancer reference value of 0.48 ppm was derived based on mouse lung tumors. The risk characterization for ethylbenzene indicated that even the most highly exposed children and prospective parents are not at risk for non-cancer or cancer effects of ethylbenzene.     

Inhalation pharmacokinetics: evaluating systemic extraction, total in vivo metabolism, and the time course of enzyme induction for inhaled styrene in rats based on arterial blood:inhaled air concentration ratios

A method is described for evaluating systemic extraction of soluble vapors during inhalation exposures. The physiological basis of the method is the inability to achieve complete equilibrium of vapor between arterial blood and inhaled air whenever there is substantial extraction of the soluble vapor during a single pass through the systemic circulation. The technique was applied to estimate styrene extraction ratios at the end of 6-hr exposures in male rats exposed to various concentrations of inhaled styrene. From extraction ratios and several physiological constants, metabolic constants were evaluated for styrene metabolism in vivo. In naive rats, the maximum velocity of metabolism was 10.0 mg/kg/hr, and Km was of the order of 0.2 mg/liter. Pretreatment with pyrazole (320 mg/kg, 1/2 hr before exposure) essentially abolished in vivo styrene metabolism, while pretreatment with phenobarbital (80 mg/kg/day for the 4 days before styrene exposure) increased Vmax about sixfold. Prior exposure to styrene (1000 ppm for 6 hr/day on each of 4 days before experimentation) increased Vmax by a factor of 2. Significant induction of styrene metabolism in vivo was observed in 24-hr continuous exposure to 400, 600, or 1200 ppm. A curve fitting routine was employed with a physiological model of styrene inhalation kinetics to estimate the dynamics of the induction process in the 24-hr exposures. At 400 ppm, induction began after a lag of 15.5 hr, had a half-life of 3.5 hr, and reached 2.7 times the Vmax in naive rats. At 600 ppm, it began after 10.6 hr, proceeded with a half-life of 2.2 hr, and increased Vmax by 3.4 times. At 1200 ppm, induction began earlier, 4.6 hr, and reached a greater value, 4.4 times Vmax, but had a half-life similar to that at 600 ppm. No induction occurred in 48-hr exposure to 200 ppm. Induction complicates kinetic modeling of continuous inhalation with soluble, well-metabolized vapors because it is time and concentration dependent. These methods should prove useful for studying the in vivo metabolism of other soluble, well-metabolized vapors and for examining the time course of induction of the metabolizing enzymes for these chemicals.